Artificial Polyploidy Improves Bacterial Single Cell Genome Recovery
Artificial Polyploidy Improves Bacterial Single Cell Genome Recovery
复制标题
DOI：
10.1371/journal.pone.0037387
复制
发表时间：
2012-05-22
期刊：
影响因子：
3.7
通讯作者：
Han, Cliff S.
中科院分区：
文献类型：
--
作者：
Dichosa, Armand E. K.;Fitzsimons, Michael S.;Han, Cliff S.
背景资料：单细胞基因组学（SCG）是一种方法的组合，其目标是从单细胞中破译完整的基因组序列，主要应用于基因组较小的生物体，如细菌和古细菌。先前的单细胞研究表明，可以获得基因组的重要部分。然而，基因组DNA的断裂和扩增偏倚使得用单细胞获得完整基因组变得非常具有挑战性。我们研究了一种人工的方法来诱导多倍体在枯草芽孢杆菌ATCC 6633通过阻断细胞分裂，并已表明，我们可以显着提高性能的基因组测序从一个单一cell.Methodology/主要发现：我们抑制细菌细胞骨架蛋白FtsZ在B。枯草芽孢杆菌与FtsZ抑制化合物PC 190723，导致较大的未分裂的单细胞与其基因组的多个拷贝。这些较大的分选细胞的qPCR检测显示出更高的DNA含量，具有更小的扩增偏差，和更大的基因组recovery比untreated cells.Significance：这里提出的方法显示出从单个细菌细胞获得几乎完整的基因组序列的潜力。自然界中有数百万未培养的细菌物种，这种方法具有巨大的希望，可以深入了解尚未发现的物种的基因组新奇，并且考虑到人工多倍性的暂时影响，以及分选和区分细胞大小和基因组DNA含量差异的能力，可以允许回收特定生物体及其基因组。
Background: Single cell genomics (SCG) is a combination of methods whose goal is to decipher the complete genomic sequence from a single cell and has been applied mostly to organisms with smaller genomes, such as bacteria and archaea. Prior single cell studies showed that a significant portion of a genome could be obtained. However, breakages of genomic DNA and amplification bias have made it very challenging to acquire a complete genome with single cells. We investigated an artificial method to induce polyploidy in Bacillus subtilis ATCC 6633 by blocking cell division and have shown that we can significantly improve the performance of genomic sequencing from a single cell.Methodology/Principal Findings: We inhibited the bacterial cytoskeleton protein FtsZ in B. subtilis with an FtsZ-inhibiting compound, PC190723, resulting in larger undivided single cells with multiple copies of its genome. qPCR assays of these larger, sorted cells showed higher DNA content, have less amplification bias, and greater genomic recovery than untreated cells.Significance: The method presented here shows the potential to obtain a nearly complete genome sequence from a single bacterial cell. With millions of uncultured bacterial species in nature, this method holds tremendous promise to provide insight into the genomic novelty of yet-to-be discovered species, and given the temporary effects of artificial polyploidy coupled with the ability to sort and distinguish differences in cell size and genomic DNA content, may allow recovery of specific organisms in addition to their genomes.